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基因打靶技术可以定向改变细胞或生物遗传信息,正逐步成为功能基因组学研究的重要分子生物学手段。其中,类转录激活因子效应物核酸酶(transcription activator-like effector nuclease,TALEN)技术已在多个农作物中实现基因的定点修饰,其已成为作物分子育种一种有效的技术手段。本研究利用TALEN技术,将水稻(Oryza sativa)基因组中已知的苯达松(bentazone)抗性基因CYP81A6进行定点突变,以创制苯达松敏感突变体育种材料。通过农杆菌(Agrobacterium tumefaciens)介导转化粳稻品种武运粳7号(Oryza sativa subsp.japonica‘Wuyunjing 7’)愈伤,共获得110株T0水稻阳性转化苗,利用改良的PCR模板快速制备方法并结合高分辨率溶解曲线(high resolution melting,HRM)检测技术筛选T0代阳性植株,获得3株CYP81A6基因突变体材料,定点突变效率约为2.73%。基因测序分析及田间测试结果表明3株材料存在不同形式的基因序列变异,且均为嵌合体材料。对T1代群体继续进行苯达松敏感测试,发现该群体仍会出现大量的苯达松敏感植株,但未能筛选到纯合突变体。本研究虽未能获得稳定遗传的苯达松敏感基因纯合水稻突变体材料,但为下一步优化试验方案及继续筛选突变体积累了丰富的经验。
Gene targeting technology can be targeted to change the genetic information of cells or organisms, is gradually becoming an important molecular biology means of functional genomics. Among them, the transcription activator-like effector nuclease (TALEN) technology has been used to perform site-directed gene modification in a variety of crops and has become an effective technical means for crop molecular breeding. In this study, the known bentazone resistance gene CYP81A6 in rice (Oryza sativa) genome was mutated by TALEN technique to create the bentasone sensitive mutant breeding material. A total of 110 T0 rice positive transformants were obtained by Agrobacterium tumefaciens-mediated transformation of a japonica rice, Oryza sativa subsp. Japonica’Wuyunjing 7 ’, and the rapid PCR method was used to prepare T0 generation positive plants were screened by high resolution melting (HRM) detection. Three CYP81A6 gene mutants were obtained. The site-directed mutagenesis efficiency was about 2.73%. Gene sequencing analysis and field test results showed that there were different forms of gene sequence variation among the three materials, and all were chimera materials. Bentazon susceptible test was continued on T1 generation and found that there was still a large number of bentazon sensitive plants in this population, but homozygous mutants could not be screened out. Although we did not get the stable transgenic bentazon susceptible gene homozygous rice mutant, we accumulated rich experience in the next step to optimize the experimental program and continue screening the mutants.